The energy-transducing ATPase complex in membranes of Escherichia coli functions as a reversible ATP driven proton pump. The complex is composed of two distinct entities: The F1 enzymatic activity that catalyzes hydrolysis of ATP and an intrinsic membrane sector, Fo, which promotes the translocation of protons across the membrane. The primary goal of this research project is to define the polypeptide components of the Fo proton-translocase, and to reconstitute the activity from purified protein constituents in phospholipid liposomes. Much of the rationale for the experimental approach taken is based upon the fact that either ATP-driven proton-translocation by F1-Fo complexes or passive proton-translocation by Fo can be inhibited by carbodiimides. Direct evidence indicates that dicyclohexylcarbodiimide (DCCD) blocks these proton-translocating reactions by covalently modifying a specific proteolipid component of Fo. Our present approach involves: (1) characterization of the carbodiimide-reactive proteolipid of Fo and assessment of whether it alone catalyzes proton-translocation, (2) purification of the entire F1-Fo complex in order to define the subunits of the Fo sector, (3) isolation of mutants with defective proton-translocating Fo sectors so that the polypeptides of Fo that are essential for proton-translocation can be identified.